Multifunctional coupling reagents having an azlactone function

ABSTRACT

Novel compounds, having an azlactone function, of formula (I), to be used as multifunctional coupling agents, and a method for coupling a biomolecule and a target molecule using such a compound are described. A diagnosis reagent, a kit for implementing the coupling method, a method for separating, detecting and/or characterizing at least one molecule of interest, and a composition including a novel compound are also described.

A subject of the present invention is novel multifunctional compounds,and their use in click chemistry in a method for coupling a biomoleculeand a target molecule.

The field of the invention is the design and the synthesis of moleculeshaving at least two distinct reactive chemical functions making itpossible to carry out chemistry reactions called “click” reactionsbetween biomolecules containing primary amine groups such as proteins,nucleic acids or certain polysaccharides.

Currently a very high demand exists in the field of bioconjugation orligation of biomolecules with natural or synthetic polymers, because ofthe potential applications of such bioconjugates in biology,biochemistry, biotechnologies and nanomedicine.

The immense complexity and diversity of life represents an enormouschallenge for the scientists trying to discover its chemical base. Thedecoding of the genetic composition of different organisms is notentirely useful if it is not accompanied by knowledge of the function ofthe encoded proteins. Bioconjugation which consists of coupling twobiomolecules by a covalent bond is a means of achieving this goal.

In particular, bioconjugation represents a useful approach forunderstanding the regulation and the biological function of certainproteins and certain biopolymers by the binding of ligands. Thisapproach consists of attaching small synthetic or natural molecules,which can function like probes, to the molecules of biological interestin order to monitor the binding of the ligand. Such probes include forexample fluorescent molecules, biotin and Nuclear Magnetic Resonance(NMR) probes. This technique offers the possibility of rapidly testing alarge number of potential ligands. Another approach consists ofintroducing synthetic functional groups onto biomolecules, a step whichis followed by their immobilization on surfaces by a chemoselectivereaction. The immobilized biomolecule can then be exposed to differentmolecules in order to identify its ligands. DNA chips and protein chipsare common examples of such an approach.

Bioconjugation also allows biochemical tests, diagnostic applications bythe qualitative and quantitative detection of analytes in clinicalsamples, applications in the field of in vivo imaging, for example bythe binding of contrast agents conjugated to antibodies, and the use ofimmobilized enzymes used as industrial catalysts. Other less commonmolecules used in bioconjugation are oligosaccharides, syntheticpolymers such as polyethyleneglycol (PEG), and carbon nanotubes.

The difficulty to be overcome in the bioconjugation reactions is theloss of the biological function of the target molecule due to poorcontrol of the site where the modification takes place. The methods ofbioconjugation developed recently are more specific to a site andinvolve minimal disturbances to the active form of the biomolecule.Moreover, certain immobilized biomolecules can exhibit an increasedligand-binding ability.

The most common chemical bioconjugation methods are based on cysteine orlysine residues. More recent methods also use synthetic functionalgroups, such as olefins.

The derivatization of proteins by a thiolate group of a cysteine residueis a common method of bioconjugation, as the thiolates are potentialnucleophiles in an aqueous solution. Thiol-reactive functional groupsinclude the iodoacetamides, maleimides and disulphides. By way ofexample, the iodoacetamides are used in a quite standard fashion intests for determining of the presence of free cysteines in proteins.More recently, iodoacetamide groups have been used for labellingproteins with fluorophores, or for immobilizing proteins.

As the amide bonds have a high stability, they are the targets of choicefor bioconjugation. For example, a protein can be treated with a smallmolecule or surface having an activated ester bond in order to form anamide bond with the amine groups of the lysines and the N-terminal ends.Native chemical ligation and Staudinger ligation are two recentapproaches for generating amide bonds at specific sites of a givenprotein.

Another target for bioconjugation is the easy synthesis ofcarbon-nitrogen double bonds by condensation of nitrogen-containingbases with aldehydes or ketones in aqueous solutions at neutral pH. Inthis way oximes (C═N—O) and hydrazones (C═N—N) can be synthesized whichare more stable than simple imines (C═N). Thus, the glucides are capableof being modified by carbon-nitrogen double bonds because their hydroxylgroups can be easily oxidized to aldehydes. Alternatively, ketones canbe introduced onto sugars present on the surface of the cells bybiosynthesis. The sugars immobilized by the oxime bonds have been usedto produce sugar chips. Numerous examples of oligonucleotides conjugatedby oxime or hydrazone bonds are given in the prior art. As anapplication, the use of peptide chips by the immobilization of peptidesmaking it possible to detect antibodies in blood samples can bementioned.

The Huisgen cycloaddition reaction in the presence of a copper Cu(I)catalyst is one the most used in bioconjugation. It consists of thereaction between a terminal alkyne and an azide generating a1,4-disubstituted triazole. This reaction has been used for very manyapplications, in particular the labelling of proteins with smallmolecules, the immobilization of proteins and of peptides, applicationsin proteomics, the immobilization of sugars, the functionalization ofDNA, and the binding of fluorescent molecules onto viruses and bioactivepolymers.

In the last few years, the use of functionalized polymers with anazlactone (or oxazolone) function for the development of functionalmaterials has increased. Due to the fact that the azlactones can reactby ring opening reactions with a wide diversity of nucleophilic species,such as the primary amines, hydroxyl groups, and thiol functions,materials functionalized with an azlactone function can serve asreactive platforms for the post-synthesis or post-productionintroduction of a wide range of chemical functions into solublepolymers, insoluble supports and surfaces. The reactivity of thiselectrophilic ring is such that an opening reaction leads to theformation of a functional linker which is not very reactive. In fact, itis constituted either by two amide bonds if the nucleophile is a primaryamine, or one amide bond and one ester bond if the nucleophile is analcohol, or one amide bond and one thioester bond if the nucleophile isa thiol. They frame a highly hindered tetrasubstituted centre. Theoverall site constituting the linker is therefore not very reactive.

The last decade has seen a significant increase in both the number andthe variety of applications exploiting the properties and the reactivityof such functionalized polymers. Various studies have shown theusefulness of supports having an azlactone function such as solublesupports, films, monoliths and insoluble supports in the form of beads.The applications of these supports, related to the composition and themorphology of the support, however remain limited and mainly amount tothe immobilization of enzymes, catalysts, ligands or to trapping amines.

For example, concerning the monoliths, two possible applications, inconnection with their structure, have been highlighted: the hydrophilicmonoliths are used in an aqueous medium in order to immobilize enzymessuch as trypsine or bovine serum albumin (BSA) and the hydrophobicmonoliths are used for trapping amines in an organic medium. In the sameway as for the monoliths, the insoluble cross-linked supports having anazlactone functionality in the form of beads have two main applications:the immobilization of enzymes and the trapping of amines. Thus, SOLA J.et al. (Angew. Chem. Int. Ed., 2010, 49, 6836-6839) disclose azides thatare used for studying modifications to the helical structures ofpeptidomimetic oligomers based on aminoisobutyric acid (Aib).International application WO 2004/081538 describes azlactones which areused as intermediates for the synthesis of poly (oxazolone) homopolymerscapable of being conjugated with different active agents andinternational application WO 93/25594 describes azlactone groups fixedon supports and used for fixing the active agents.

In current therapeutic research, novel methodologies have been developedin order to more rapidly access a wide diversity of compounds. Inparticular, chemical ligation by “click” chemistry has been proposed bySharpless in 2001 (H. C. Kolb, M. G. Finn and K. B. Sharpless (2001).“Click Chemistry: Diverse Chemical Function from a Few Good Reactions”.Angewandte Chemie International Edition 40 (11): 2004-2021) in order togenerate different original structures of standard pharmacophores. Areaction can be considered as a “click” reaction if it corresponds tothe following criteria:

-   -   modularity    -   stereoselectivity    -   insensitivity to oxygen and to water    -   high purity and yield.

“Click” chemistry therefore makes it possible to develop a set ofclear-cut and modular reactions which can be implemented under mildconditions, without tedious purification, without formation ofby-products, having a wide spectrum of substrates which arephysiologically stable and/or compatible with biological media and leadto atom economy at high yields. The main “click” reactions consist offorming very energy-efficient carbon-heteroatom bonds, in particular aring opening nucleophilic reaction or a cycloaddition reaction. A typeof reaction which is widely represented in “click” chemistry is theabovementioned alkyne-azide cycloaddition catalyzed with Cu(I). When the“click” reactions are compatible with the chemical functions and thebiological media, whether in vitro or in vivo, these reactions arecalled bio-orthogonal.

Although numerous reagents capable of being used in these clickchemistry reactions exist, a need remains for novel reagents allowingefficient and rapid reactions in an aqueous medium, without theformation of by-products which are difficult to remove and/or are toxic.

A purpose of the invention is therefore to propose a family of novelmultifunctional reactive coupling agents which can be used for thecombination, by covalent bonds, of biomolecules such as proteins,peptides, DNA, certain polysaccharides, with one or more otherbiomolecule(s), a naturel or synthetic polymer, or a reactive surface.These novel compounds are of quite particular interest for “click”chemistry applied to biology and chemical synthesis.

Another purpose of the invention is also to overcome the drawbacks ofthe state of the art by proposing novel coupling reagents:

allowing combinations by bio-orthogonal methods, both in vitro and invivo,

the reactive functionalities of which are also orthogonal betweenthemselves,

the high reactivity of which is compatible with biological media,

having a resistance to hydrolysis in an aqueous medium,

without the formation of by-products which are toxic or difficult toremove,

applicable to a wide range of biological or synthetic macromolecules,

leading to chemical bonds which are robust and compatible with a vastrange of chemical functionalities as well as with biological media andliving organisms, in vitro and in vivo.

Certain compounds corresponding to general formula (I)

in which

R₁ and R₂ represent independently of one another a (C₁-C₁₀)alkyl group,a (C₂-C₆)cycloalkyl group, an aryl group, an aryl(C₁-C₁₀)alkyl group, ora heterocyclic group,

R₃ and R₄ represent independently of one another a hydrogen atom, a(C₁-C₁₀)alkyl group, an aryl group or an aryl(C₁-C₁₀)alkyl group, and

Y represents a reactive function which can be activated by clickchemistry, selected from the group comprising the azides, alkynes,cycloalkynes and conjugated dienes,

are known as such but are not described as multifunctional couplingreactive agents (see SOLA J. cited previously).

According to the present invention, the term “(C₁-C₁₀)alkyl” representsa linear or branched, saturated hydrocarbon-containing group, having 1to 10 carbon atoms, advantageously from 1 to 5 carbon atoms. By way ofexample, the methyl, ethyl, propyl, isopropyl, butyl, isobutyl,sec-butyl, tertiobutyl, pentyl, neopentyl, isopentyl, tert-pentyl,hexyl, isohexyl, heptyl, octyl, nonyl and decyl groups can be mentioned.

The term “aryl” represents a mono- or bicyclic, aromatichydrocarbon-containing group comprising from 6 to 10 carbon atoms. Byway of example the phenyl and naphthyl groups can be mentioned.

By the term “aryl(C₁-C₁₀)alkyl”, is meant an alkyl group having 1 to 10carbon atoms, advantageously from 1 to 5 carbon atoms as definedpreviously and containing an aryl group as defined above.

The term “heterocyclic group” represents any saturated or unsaturatedheterocycle, comprising from 3 to 7 carbon atoms and containing 1 to 3heteroatoms selected from the group constituted by oxygen, nitrogen orsulphur. For example, the piperidinyl, pyrrolidinyl, piperazinyl,pyridyl, piridinyl, imidazolyl, furyl, morpholinyl, oxetanyl,tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothienyl, and thiazolylgroups can be mentioned.

The term “azides” represents the salts of hydrozoic acid HN₃, as well asthe organic azides in which one of the nitrogen atoms is covalentlybound to a carbon atom of an organic compound. As examples methyl azideand phenyl azide can be mentioned.

The term “alkynes” represents hydrocarbons having an unsaturationcharacterized by the presence of a triple carbon-carbon bond. Asexamples ethyne, propyne, but-1-yne, and but-2-yne can be mentioned.

By “cycloalkynes” is meant any ring of carbon atoms containing one ormore triples bonds, such as cyclooctyne.

The term “conjugated dienes” represents hydrocarbons having two doublebonds separated by one single bond. By way of example, butadiene,isoprene, 2,3-dimethyl-1,3-butadiene, 1,3-pentadiene,2-phenyl-1,3-butadiene and furan can be mentioned.

The first chemical function common to the compounds corresponding toformula (I) used according to the invention is of azlactone(2-oxazolin-5-one) type. This group is known for reacting by ringopening with primary amine functions without requiring a catalyst andwith the formation of a robust amide bond under mild conditions, inparticular in an aqueous solution, therefore compatible with biologicalmedia. The major use of this chemical function compared to the systemsalready described resides, in addition to its intrinsic reactivityleading to a rapid and efficient reaction in an aqueous medium due toits resistance to hydrolysis, in the fact that the reaction with anamine does not lead to the formation of reaction by-products which arepossibly toxic and difficult to remove from the reaction medium. Theprimary amine functions are widespread in most biological molecules(proteins, nucleic acids in particular) and molecules of biologicalinterest, which ensures a very broad field of application for thereagents of this invention. In terms of cost, the absence of by-productsgenerated by the reaction of the azlactone group also leads to atomeconomy. Moreover, the possibility of working in an aqueous medium is anadvantage in terms of the environment.

The other common function present in the compounds corresponding toformula (I) used according to the invention corresponds to a groupcapable of participating in a cycloaddition reaction, in particular theazide, alkyne, cycloalkyne and conjugated diene groups. Thecycloaddition reactions constitute the most widespread examples of clickchemistry, in particular the Huisgen reaction mentioned previously,[4+2] cycloaddition (Diels-Alder reaction), and heterocycloaddition.These reactions are bio-orthogonal, i.e. they are compatible with thespecies present in biological media. In fact, they utilize functionalgroups which are in general neither present, nor capable of reactingwith the groups which exist within the biomolecules.

The two chemical functions present in the compounds according to theinvention are moreover orthogonal between themselves, i.e. they reactunder different conditions, independently of one another. It istherefore possible to trigger the reaction of one of these two groupswithout the other being converted, thus allowing successivechemoselective reactions.

A subject of the present invention is compounds of formula (I)

in which

R₁ and R₂ represent independently of one another a (C₁-C₁₀)alkyl group,a (C₂-C₆)cycloalkyl group, an aryl group, an aryl(C₁-C₁₀)alkyl group, ora heterocyclic group,

R₃ and R₄ represent independently of one another a hydrogen atom, a(C₁-C₁₀)alkyl group, an aryl group or an aryl(C₁-C₁₀)alkyl group, and

Y represents:

(i) an N₃ group, or(ii) a (C₁-C₁₀)alkyl-N₃ group, or(iii) an aryl-N₃ group, or(iv) an aryl(C₁-C₁₀)alkyl-N₃ group, or(v) a (C₁-C₁₀)alkyl-C≡C—R₅ group, or(vi) an aryl-C≡C—R₅ group, or(vii) an aryl(C₁-C₁₀)alkyl-C≡C—R₅ group, or(viii) an —O—C(O)—(CH₂)_(n)—C≡CR₅ group, with n an integer comprisedbetween 1 and 10, in particular an —O—C(O)—(CH₂)_(n)—C≡CH group, with ncomprised between 1 and 5,

with R₅ representing

-   -   (a) either a hydrogen atom,    -   (b) or a (C₁-C₁₀)alkyl group,    -   (c) or an aryl group,    -   (d) or an aryl(C₁-C₁₀)alkyl group,    -   all these groups being able to contain two conjugated double        bonds, optionally in a ring,    -   (e) or a protective group of the triple bond.

In the definitions (i) to (viii), the terms (C₁-C₁₀)alkyl, aryl andaryl(C₁-C₁₀)alkyl refer to the definitions given previously.

As examples of a (C₁-C₁₀)alkyl, aryl or aryl(C₁-C₁₀)alkyl group, capableof containing two conjugated double bonds, optionally in a ring, inparticular 1,3-butadienyl, cyclopentadienyl, pyrrolyl and furyl can bementioned.

All the groups mentioned have in common the ability to produce a [3+2]dipolar cycloaddition or a cycloaddition or a [4+2] heterocycloadditionreaction.

The protective groups of the triple bond are well known to a personskilled in the art. As examples, the trialkysilyls, in particulartrimethylsilyl, triethylsilyl, tert-butyldimethylsilyl andbenzyldimethylsilyl can be mentioned.

According to another advantageous embodiment of the invention, thecompounds of formula (I) are those for which R₁ and R₂ each represent a(C₁-C₁₀)alkyl group, R₃ represents a hydrogen atom, R₄ represents amethyl group, and Y represents either an N₃ group, or an—O—C(O)—(CH₂)₃—C≡CH group.

Advantageously, the R₁, R₂ and R₄ groups each represent a methyl group,R₃ a hydrogen atom and Y an N₃ group.

According to another advantageous embodiment of the invention, R₁, R₂and R₄ each refer to a methyl group, R₃ a hydrogen atom and Y an—O—C(O)—(CH₂)₃—C≡CH group.

Another subject of the invention is a method for coupling a biomoleculeand a target molecule selected from the group comprising a molecule ofbiological interest, a naturel or synthetic polymer, and a reactivesurface, said method utilizing a compound of formula (I) in which

R₁ and R₂ represent independently of one another a (C₁-C₁₀)alkyl group,a (C₂-C₆)cycloalkyl group, an aryl group, an aryl(C₁-C₁₀)alkyl group, ora heterocyclic group,

R₃ and R₄ represent independently of one another a hydrogen atom, a(C₁-C₁₀)alkyl group, an aryl group or an aryl(C₁-C₁₀)alkyl group, and

Y represents a reactive function which can be activated by clickchemistry, selected from the group comprising the azides, alkynes,cycloalkynes and conjugated dienes,

and comprising the following steps:

-   -   bringing said compound of formula (I) into contact with a        biomolecule,    -   bringing said compound of formula (I) bound to the biomolecule        into contact with the target molecule and if necessary,    -   isolating the coupling product.

In an advantageous embodiment of the invention, the coupling methodutilizes a compound of formula (I) in which

R₁ and R₂ represent independently of one another a (C₁-C₁₀)alkyl group,a (C₂-C₆)cycloalkyl group, an aryl group, an aryl(C₁-C₁₀)alkyl group, ora heterocyclic group,

R₃ and R₄ represent independently of one another a hydrogen atom, a(C₁-C₁₀)alkyl group, an aryl group or an aryl(C₁-C₁₀)alkyl group, and

Y represents:

(i) an N₃ group, or(ii) a (C₁-C₁₀)alkyl-N₃ group, or(iii) an aryl-N₃ group, or(iv) an aryl(C₁-C₁₀)alkyl-N₃ group, or(v) a (C₁-C₁₀)alkyl-C≡C—R₅ group, or(vi) an aryl-C≡C—R₅ group, or(vii) an aryl(C₁-C₁₀)alkyl-C≡C—R₅ group, or(viii) an —O—C(O)—(CH₂)_(n)—C≡CR₅ group, with n an integer comprisedbetween 1 and 10, in particular an —O—C(O)—(CH₂)_(n)—C≡CH group, with ncomprised between 1 and 5,

with R₅ representing

-   -   (a) either a hydrogen atom,    -   (b) or a (C₁-C₁₀)alkyl group,    -   (c) or an aryl group,    -   (d) or an aryl(C₁-C₁₀)alkyl group,    -   all these groups being able to contain two conjugated double        bonds, optionally in a ring,    -   (e) or a protective group of the triple bond.

By “reactive surface”, is meant an organic or inorganic material havingchemical functions on its surface capable of reacting with the moleculesof the present invention. For example, a surface (film, fabric, etc.) ofpolypropylene or polyethylene having amine functions on the surface canbe mentioned.

According to an advantageous embodiment of the method according to theinvention, the biomolecule(s) involved in said coupling method areselected from the group comprising proteins, peptides, DNA, biologicalmarkers, hormones, vitamins, antibodies, polyamines, monosaccharides,oligosaccharides and polysaccharides, and the molecules of biologicalinterest of medicinal type such as anticancers, anti-virals, or labelssuch as fluorescent or radioactive labels.

Another subject of the invention is a diagnostic reagent utilizing atleast one compound of formula (I), in particular a compound for which

R₁ and R₂ represent independently of one another a (C₁-C₁₀)alkyl group,a (C₂-C₆)cycloalkyl group, an aryl group, an aryl(C₁-C₁₀)alkyl group, ora heterocyclic group,

R₃ and R₄ represent independently of one another a hydrogen atom, a(C₁-C₁₀)alkyl group, an aryl group or an aryl(C₁-C₁₀)alkyl group, and

Y represents:

(i) an N₃ group, or(ii) a (C₁-C₁₀)alkyl-N₃ group, or(iii) an aryl-N₃ group, or(iv) an aryl(C₁-C₁₀)alkyl-N₃ group, or(v) a (C₁-C₁₀)alkyl-C≡C—R₅ group, or(vi) an aryl-C≡C—R₅ group, or(vii) an aryl(C₁-C₁₀)alkyl-C≡C—R₅ group, or(viii) an —O—C(O)—(CH₂)_(n)—C≡CR₅ group, with n an integer comprisedbetween 1 and 10, in particular a —O—C(O)—(CH₂)_(n)—C≡CH group, with ncomprised between 1 and 5,

with R₅ representing

-   -   (a) either a hydrogen atom,    -   (b) or a (C₁-C₁₀)alkyl group,    -   (c) or an aryl group,    -   (d) or an aryl(C₁-C₁₀)alkyl group,    -   all these groups being able to contain two conjugated double        bonds, optionally in a ring,    -   (e) or a protective group of the triple bond.

A subject of the invention is also the use of the compounds of formula(I) in which

R₁ and R₂ represent independently of one another a (C₁-C₁₀)alkyl group,a (C₂-C₆)cycloalkyl group, an aryl group, an aryl(C₁-C₁₀)alkyl group, ora heterocyclic group,

R₃ and R₄ represent independently of one another a hydrogen atom, a(C₁-C₁₀)alkyl group, an aryl group or an aryl(C₁-C₁₀)alkyl group, and

Y represents a reactive function which can be activated by clickchemistry, selected from the group comprising the azides, alkynes,cycloalkynes and conjugated dienes,

as a diagnostic reagent and a diagnostic method utilizing the set of thecompounds of formula (I).

In fact, the coupling agents are of use in the field of diagnostics, inparticular in the detection of reactions of the ligand-anti-ligand typesuch as antigen-antibody or protein-protein, as they make it possible todirectly couple a molecule of biological interest, such as an antigen,to a molecule called a development molecule, such as an enzyme. The bondof the biological molecule of interest to another molecule, such as anantibody, is demonstrated due to the development molecule. It can bealso useful for coupling a fluorescent probe to a molecule of biologicalinterest for its detection by functional imaging.

Another subject of the invention is a kit for the implementation of acoupling and bioconjugation method comprising at least one compound offormula (I), in particular those for which

R₁ and R₂ represent independently of one another a (C₁-C₁₀)alkyl group,a (C₂-C₆)cycloalkyl group, an aryl group, an aryl(C₁-C₁₀)alkyl group, ora heterocyclic group,

R₃ and R₄ represent independently of one another a hydrogen atom, a(C₁-C₁₀)alkyl group, an aryl group or an aryl(C₁-C₁₀)alkyl group, and

Y represents:

(i) an N₃ group, or(ii) a (C₁-C₁₀)alkyl-N₃ group, or(iii) an aryl-N₃ group, or(iv) an aryl(C₁-C₁₀)alkyl-N₃ group, or(v) a (C₁-C₁₀)alkyl-C≡C—R₅ group, or(vi) an aryl-C≡C—R₅ group, or(vii) an aryl(C₁-C₁₀)alkyl-C≡C—R₅ group, or(viii) an —O—C(O)—(CH₂)_(n)—C≡CR₅ group, with n an integer comprisedbetween 1 and 10, in particular a —O—C(O)—(CH₂)_(n)—C≡CH group, with ncomprised between 1 and 5,

with R₅ representing

-   -   (a) either a hydrogen atom,    -   (b) or a (C₁-C₁₀)alkyl group,    -   (c) or an aryl group,    -   (d) or an aryl(C₁-C₁₀)alkyl group,    -   all these groups being able to contain two conjugated double        bonds, optionally in a ring,    -   (e) or a protective group of the triple bond.

Another subject of the invention is a method for the separation,detection and/or characterization of at least one molecule of interestpotentially present in a medium, comprising at least one step ofutilizing a kit according to the invention or at least one compound offormula (I) in which

R₁ and R₂ represent independently of one another a (C₁-C₁₀)alkyl group,a (C₂-C₆)cycloalkyl group, an aryl group, an aryl(C₁-C₁₀)alkyl group, ora heterocyclic group,

R₃ and R₄ represent independently of one another a hydrogen atom, a(C₁-C₁₀)alkyl group, an aryl group or an aryl(C₁-C₁₀)alkyl group, and

Y represents a reactive function which can be activated by clickchemistry, selected from the group comprising the azides, alkynes,cycloalkynes and conjugated dienes.

Another subject of the invention is a composition comprising a compoundof formula (I) in which

R₁ and R₂ represent independently of one another a (C₁-C₁₀)alkyl group,a (C₂-C₆)cycloalkyl group, an aryl group, an aryl(C₁-C₁₀)alkyl group, ora heterocyclic group,

R₃ and R₄ represent independently of one another a hydrogen atom, a(C₁-C₁₀)alkyl group, an aryl group or an aryl(C₁-C₁₀)alkyl group, and Yrepresents:

(i) an N₃ group, or(ii) a (C₁-C₁₀)alkyl-N₃ group, or(iii) an aryl-N₃ group, or(iv) an aryl(C₁-C₁₀)alkyl-N₃ group, or(v) a (C₁-C₁₀)alkyl-C≡C—R₅ group, or(vi) an aryl-C≡C—R₅ group, or(vii) an aryl(C₁-C₁₀)alkyl-C≡C—R₅ group, or(viii) an —O—C(O)—(CH₂)_(n)—C≡CR₅ group, with n an integer comprisedbetween 1 and 10, in particular an —O—C(O)—(CH₂)_(n)—C≡CH group, with ncomprised between 1 and 5,

with R₅ representing

-   -   (a) either a hydrogen atom,    -   (b) or a (C₁-C₁₀)alkyl group,    -   (c) or an aryl group,    -   (d) or an aryl(C₁-C₁₀)alkyl group,    -   all these groups being able to contain two conjugated double        bonds, optionally in a ring,    -   (e) or a protective group of the triple bond in combination with        an aqueous or organic medium.

As organic medium, for example dimethylsulphoxide (DMSO) compatible withthe cell cultures can be mentioned.

The invention is illustrated by Examples 1 to 6 and FIG. 1 which follow.

Examples 1, 2 and 4, 5 illustrate the synthesis of compounds accordingto the invention and Examples 3 and 6 the method for coupling a compoundof the invention and a biomolecule:lysozyme.

FIG. 1 represents the MALDI-TOF analysis spectrum of thelysozyme-2-(1-azidoethyl)-4,4-dimethyloxazol-5(4H)-one conjugateobtained according to the coupling method described in Example 3.

EXAMPLE 1 2-(1-azidoethyl)-4,4-dimethyloxazol-5(4H)-one

2-(1-azidoethyl)-4,4-dimethyloxazol-5(4H)-one is prepared according toDiagram 1 below.

1.1. 2-(1-bromoethyl)-4,4-dimethyloxazol-5(4H)-one

This synthesis intermediate is prepared according to the proceduredescribed by K. M. Lewandowski et al. in the patent U.S. Pat. No.6,762,257 B1 (steps 1 and 2 of Diagram 1)

1.2. 2-(1-azidoethyl)-4,4-dimethyloxazol-5(4H)-one

2-(1-azidoethyl)-4,4-dimethyloxazol-5(4H)-one is then obtained (step 3)according to the protocol below.

In a 25-mL flask equipped with a magnetic stirrer, under an argonatmosphere, a solution of 0.65 g (0.01 mol) of sodium azide in 4 mL ofanhydrous dimethylformamide (DMF) is prepared, which is cooled downusing an ice bath. A solution comprising 2.20 g, (0.01 mol) of2-(1-bromoethyl)-4,4-dimethyloxazol-5(4H)-one, prepared in step 1.1., in2 mL of anhydrous DMF is poured dropwise into this mixture and leftunder stirring at 0° C. for 2 h. The mixture is left to return toambient temperature under stirring for 24 h. The solvent is evaporatedoff under reduced pressure and the residue is taken up in ethyl acetatethen the solution is filtered in order to remove the sodium bromide. Thefiltrate is poured into a separating funnel and the solution is washedwith dilute HCl (5%) then saturated aqueous NaHCO₃. The organic phase isdried over MgSO₄, filtered and concentrated under vacuum. The yellow oilobtained is purified by silica column chromatography with, successivelyn-hexane, then an n-hexane:ethyl acetate mixture 8:2 v/v, in order toproduce an oil (0.57 g, 31%) which crystallizes when cold.

The 2-(1-azidoethyl)-4,4-dimethyloxazol-5(4H)-one is characterized byproton nuclear magnetic resonance (¹H NMR) and by carbon 13 nuclearmagnetic resonance (¹³C NMR).

¹H NMR (400 MHz, CDCl₃, δ ppm): 1.46 ppm (s, 6H, —C(CH₃)₂), 1.56 ppm (d,3H, —CH(CH₃)N₃), 4.27 ppm (t, 1H, CH(CH₃)N₃).

¹³C NMR (400 MHz, CDCl₃, δ ppm): 16.32 ppm (—C(CH₃)N₃), 24.46 ppm(C(CH₃)₂), 53.76 ppm (—C(CH₃)N₃), 65.59 ppm (—C(CH₃)₂), 161.56 ppm(C═N), 180.11 ppm (C═O).

EXAMPLE 2 1-(4,4-dimethyl-5-oxo-4,5-dihydrooxazol-2-yl)ethylhex-5-ynoate

1-(4,4-dimethyl-5-oxo-4,5-dihydrooxazol-2-yl)ethyl hex-5-ynoate isprepared according to Diagram 2 below.

2-(1-bromoethyl)-4,4-dimethyloxazol-5(4H)-one is prepared according toExample 1.1.

2.2 1-(4,4-dimethyl-5-oxo-4,5-dihydrooxazol-2-yl)ethyl hex-5-ynoate2.2.1. Caesium salt of 5-hexynoic acid

5-hexynoic acid is prepared by reaction of caesium carbonate (4.10 g,0.0126 mol) with 5-hexynoic acid (4.6 g, 0.0411 mol) in solution in DMF(6.0 mL) at ambient temperature for 16 h. The reaction mixture isfiltered, the solvent is removed under reduced pressure and the residuetaken up in diethyl ether. After filtration, the caesium salt of5-hexynoic acid (4.82 g, yield 78%) is dried under vacuum at 40° C.

2.2.2. 1-(4,4-dimethyl-5-oxo-4,5-dihydrooxazol-2-yl)ethyl hex-5-ynoate

A solution of 2-(1-bromoethyl)-4,4-dimethyloxazol-5(4H)-one (0.448 g,2.03 mmol) in DMF (2 mL) is added dropwise to a solution of caesium saltof 5-hexynoic acid (0.490 g, 2 mmol) in anhydrous DMF (3 mL) placed in aflask cooled down with an ice bath. At the end of the addition, thereaction mixture is left under stirring at 0° C. for 2 h then left toreturn to ambient temperature over 24 h. The solvent is removed underreduced pressure then the residue is taken up in ethyl acetate, filteredand concentrated under vacuum. The yellow oil obtained is purified bysilica column chromatography in order to produce 0.310 g (61%) of1-(4,4-dimethyl-5-oxo-4,5-dihydrooxazol-2-yl)ethyl hex-5-ynoate.

The 1-(4,4-dimethyl-5-oxo-4,5-dihydrooxazol-2-yl)ethyl hex-5-ynoate ischaracterized by proton nuclear magnetic resonance (¹H NMR) and bycarbon 13 nuclear magnetic resonance (¹³C NMR).

¹H NMR (400 MHz, CDCl₃, δ ppm): 1.46 ppm (s, 6H, —C(CH₃)₂), 1.56 ppm (d,3H, —CH(CH₃)OCO), 1.85 ppm (quinquet, 2H, HC≡C—CH₂CH₂CH₂—O—CO—), 2.00ppm (HC≡C—), 2.29 ppm (triplet-doublet, HC≡C—CH₂CH₂CH₂—O—CO—), 2.54 ppm(HC≡C—CH₂CH₂CH₂—O—CO—), 5.57 ppm (quadruplet, 1H, —CH₂—COOCH(CH₃)—).

¹³C NMR (400 MHz, CDCl₃, δ ppm): 17.01 ppm (—CH₂—COOCH(CH₃)), 17.75 ppm(HC≡C—CH₂—), 23.50 ppm (HC≡C—CH₂CH₂CH₂—), 24.42 ppm (C(CH₃)₂), 32.62ppm(—CH₂CH₂CO)—), 65.04 ppm (C(CH₃)₂), 65.45 ppm (HC≡C—CH₂—), 69.50 ppm(—COO—CH(CH₃)—), 83.24 ppm (HC≡C—CH₂—), 162.06 ppm (C═N), 172.26 ppm(—CH₂—COO—CH(CH₃)—), 180.52 ppm (C═O)_(ring).

EXAMPLE 3 Process for coupling2-(1-azidoethyl)-4,4-dimethyloxazol-5(4H)-one with lysozyme

The coupling of a model protein, lysozyme, with2-(1-azidoethyl)-4,4-dimethyloxazol-5(4H)-one is described below:

3.1. Procedure

In a flask equipped with a magnetic stirrer, a solution of lysozyme(114.2 mg, 8.0×10⁻⁶ mol) in dimethylsulphoxide (DMSO, 10.00 mL) isprepared, to which trimethylamine (TEA, 0.20 mL, 1.49×10⁻³ mol) isadded. After stirring for 15 minutes at ambient temperature, a solutionof 2-(1-azidoethyl)-4,4-dimethyloxazol-5(4H)-one prepared according toExample 1 (0.252 g, 1.38×10⁻³ mol) in DMSO (1.00 mL) is added and thereaction mixture is left under stirring at ambient temperature for 24 h.After 24 h, an aqueous solution of HCl (HCl, 37%, 1.0 mL per 30.0 mL ofsolution) is added then the solution is dialyzed with a dialysismembrane (cut-off threshold=MWCO=3500) against an aqueous solution ofmethanol (water:methanol=8:2 in volumes) for 24 h. The product is thenrecovered by lyophilization. The final product (0.012 g) is dissolved inDMSO (0.10 mL) for analysis by Fourier Transform infrared (FTIR)spectroscopy. Analysis by time-of-flight mass spectrometry (MALDI-TOFMS) is also carried out.

3.2. Results

The FTIR spectrum of the sample shows the presence of a new absorptionband at 2110 cm⁻¹, characteristic of the azide group introduced onto theprotein.

The reaction is also attested by time-of-flight mass spectrometry(MALDI-TOF MS) which shows that after reaction the initial lysozyme peakat m/z=14357 has disappeared in favour of a new signal centred atm/z=15812 (FIG. 1).

EXAMPLE 4 Synthesis of 1-(4,4-dimethyl-5-oxo-4,5-dihydrooxazol-2-yl)ethyl hexa-2,4-dienoate

1-(4,4-dimethyl-5-oxo-4,5-dihydrooxazol-2-yl)ethylhexa-2,4-dienoate isprepared according to the diagram below:

4.1. 2-(1-bromoethyl)-4,4-dimethyloxazol-5(4H)-one

This is prepared according to the procedure described by K. M.Lewandowski et al. in the patent U.S. Pat. No. 6,762,257 B1 (2004).

4.2. 1-(4,4-dimethyl-5-oxo-4,5-dihydrooxazol-2-yl)ethylhexa-2,4-dienoate

Potassium sorbate (1.12 g, 7.5×10⁻³ mol),2-(1-bromoethyl)-4,4-dimethyloxazol-5(4H)-one (1.68 g, 7.6×10⁻³ mol) andanhydrous DMF (20.0 mL) are introduced into a flask equipped with amagnetic stirrer. Then, the reaction mixture is stirred and heated at60° C. under argon for 17 h. The solvent (DMF) is removed under reducedpressure. The residue is taken up in acetone then filtered on frit. Thefiltrate obtained is concentrated under vacuum. The final product isobtained in the form of a dark yellow oil with a yield of 95% (1.90 g)which crystallizes when cold.

¹H NMR (400 MHz, acetone D₆, δ ppm): 1.40 ppm (6H, —C(CH₃)₂), 1.59 ppm(d, 3H, —COOCH(CH₃)), 1.90 ppm (d, 3H, H₃C—CH₂═CH₂—), 5.63 ppm (q, 1H,—CH(CH₃)OCO), 5.91 (d, 1H, CH₃—CH═CH—CH═CH—), 6.34 ppm(CH₃—CH═CH—CH═CH—), 7.34 ppm (1H, CH₃—CH═CH—CH═CH—).

¹³C NMR (400 MHz, acetone D₆, δ ppm): 17.01 ppm (—COOCH(CH₃)—), 18.51ppm (H₃C—CH₂═CH₂—), 24.21 ppm and 24.31 ppm (C(CH₃)₂), 65.04 ppm(C(CH₃)₂), 65.85 ppm (—COO—CH(CH₃)—), 118.47 (CH₃—CH═CH—CH═CH—), 130.31(CH₃—CH═CH—CH═CH—), 141.03 ppm (CH₃—CH═CH—CH═CH—), 146.88 ppm(CH₃—CH═CH—CH═CH—), 162.21 ppm (C═N), 165.92 ppm (—COO—CH(CH₃)—), 181.16ppm (C═O)_(ring).

FT-IR: v(CH)=2985-2939, v(C═O)azlactone=1826 cm⁻¹, v(C═O)ester=1720cm⁻¹, v(C═N)=1683 cm⁻¹.

EXAMPLE 5 Synthesis of2-(2-azidopropan-2-yl)-4,4-dimethyloxazol-5(4H)-one

2-(2-azidopropan-2-yl)-4,4-dimethyloxazol-5(4H)-one is synthesizedaccording to the diagram below:

5.1. 2-(2-bromopropan-2-yl)-4,4-dimethyloxazol-5(4H)-one

This intermediate is prepared according to the procedure described by K.M. Lewandowski et al. in the patent U.S. Pat. No. 6,894,133 (steps 1 and2) 5.2. 2-(2-azidopropan-2-yl)-4,4-dimethyloxazol-5(4H)-one

It is obtained (step 3) according to the following procedure:

In a 25-mL flask equipped with a magnetic stirrer, under an argonatmosphere, a solution of 0.65 g (0.01 mol) of sodium azide in 5 mL ofanhydrous DMF is prepared, which is cooled down using an ice bath. Asolution comprising 2.34 g, (0.01 mol) of2-(2-bromopropan-2-yl)-4,4-dimethyloxazol-5(4H)-one in 2 mL of anhydrousDMF is poured dropwise into this mixture. The reaction mixture isstirred at 0° C. for 2 h then at ambient temperature for 16 h. Thesolvent is evaporated off under reduced pressure. The residue is takenup in ethyl acetate then the solution is filtered in order to remove thesodium bromide. The filtrate obtained is concentrated under vacuum. Theyellow oil obtained is purified by silica column chromatography with,successively, n-hexane then an n-hexane:ethyl acetate mixture 6:4 v/v inorder to produce a colourless oil (0.84 g, 43%).

¹H NMR (200 MHz, CDCl₃, δ ppm): 1.44 ppm (s, 6H, —C(CH₃)₂), 1.56 ppm (s,6H, N₃C(CH₃)₂).

¹³C NMR (400 MHz, CDCl₃, δ ppm): 24.01 ppm (N₃C(CH₃)₂), 24.58 ppm(—C(CH₃)₂), 59.26 ppm (N₃C(CH₃)₂), 65.83 ppm (—C(CH₃)₂), 163.88 ppm(C═N), 180.41 ppm (C═O).

EXAMPLE 6 Process for coupling1-(4,4-dimethyl-5-oxo-4,5-dihydrooxazol-2-yl)ethyl hex-5-ynoate withlysozyme

In a flask equipped with a magnetic stirrer, a solution of lysozyme(228.4 mg, 16.0×10⁻⁶ mol) in dimethylsulphoxide (DMSO, 20.00 mL) isprepared, to which triethylamine (TEA, 0.30 g, 3.0×10⁻³ mol) is added.After stirring for 30 min at ambient temperature, a solution of1-(4,4-dimethyl-5-oxo-4,5-dihydrooxazol-2-yl)ethyl hex-5-ynoate (0.695g, 3.0×10⁻³ mol) in DMSO (4.00 mL) is added and the reaction mixture isstirred at ambient temperature for 24 h. After 24 h, a mixture ofconcentrated HCl (37%, 2 mL) and 90 mL of pure water is added, then thesolution is dialyzed with a dialysis membrane (cut-offthreshold=MWCO=3500) against an aqueous solution of methanol(water:methanol=8:2 in volumes) for 24 h. The product is then recoveredby lyophilization.

The final product is analyzed by time-of-flight mass spectrometry(MALDI-TOF MS). The MALDI-TOF analysis result shows that after thereaction the initial lysozyme peak at m/z=14357 has disappeared infavour of a new signal centred at m/z=17819.

1. Compound corresponding to general formula (I)

in which R₁ and R₂ represent independently of one another a(C₁-C₁₀)alkyl group, a (C₂-C₆)cycloalkyl group, an aryl group, anaryl(C₁-C₁₀)alkyl group, or a heterocyclic group, R₃ and R₄ representindependently of one another a hydrogen atom, a (C₁-C₁₀)alkyl group, anaryl group or an aryl(C₁-C₁₀)alkyl group, and Y represents: (i) an N₃group, or (ii) a (C₁-C₁₀)alkyl-N₃ group, or (iii) an aryl-N₃ group, or(iv) an aryl(C₁-C₁₀)alkyl-N₃ group, or (v) a (C₁-C₁₀)alkyl-C≡C—R₅ group,or (vi) an aryl-C≡C—R₅ group, or (vii) an aryl(C₁-C₁₀)alkyl-C≡C—R₅group, or (viii) an —O—C(O)—(CH₂)_(n)—C≡CR₅ group, with n an integercomprised between 1 and 10, in particular a —O—C(O)—(CH₂)_(n)—C≡CHgroup, with n comprised between 1 and 5, with R₅ representing (a) eithera hydrogen atom, (b) or a (C₁-C₁₀)alkyl group, (c) or an aryl group, (d)or an aryl(C₁-C₁₀)alkyl group, all these groups being able to containtwo conjugated double bonds, optionally in a ring, (e) or a protectivegroup of the triple bond.
 2. Compound according to claim 1 in which R₁and R₂ each represent a (C₁-C₁₀)alkyl group, R₃ represents a hydrogenatom, R₄ represents a methyl group, and Y represents either an N₃ group,or an —O—C(O)—(CH₂)₃—C≡CH group.
 3. Compound according to claim 2 inwhich R₁, R₂ and R₄ each represent a methyl group, R₃ a hydrogen atomand Y an N₃ group.
 4. Compound according to claim 2 in which R₁, R₂ andR₄ each denote a methyl group, R₃ a hydrogen atom and Y an—O—C(O)—(CH₂)₃—C≡CH group.
 5. Method for coupling a biomolecule and atarget molecule selected from the group comprising a molecule ofbiological interest, a naturel or synthetic polymer, and a reactivesurface, characterized in that it utilizes a compound of formula (I)

in which R₁ and R₂ represent independently of one another a(C₁-C₁₀)alkyl group, a (C₂-C₆)cycloalkyl group, an aryl group, anaryl(C₁-C₁₀)alkyl group, or a heterocyclic group, R₃ and R₄ representindependently of one another a hydrogen atom, a (C₁-C₁₀)alkyl group, anaryl group or an aryl(C₁-C₁₀)alkyl group, and Y represents a reactivefunction which can be activated by click chemistry, selected from thegroup comprising the azides, alkynes, cycloalkynes and conjugateddienes, in particular a compound according to claim 1 and in that itcomprises the following steps: bringing said compound into contact witha biomolecule, bringing said compound bound to the biomolecule intocontact with the target molecule and if necessary, isolating thecoupling product.
 6. Method according to claim 5 characterized in thatthe biomolecule(s) involved in said coupling method are selected fromthe group comprising proteins, peptides, DNA, biological markers,hormones, vitamins, antibodies, polyamines, monosaccharides,oligosaccharides and polysaccharides, and molecules of biologicalinterest of medicinal or label type.
 7. Diagnostic reagent characterizedin that it utilizes at least one compound according to claim
 1. 8.Method of using a compound of formula (I) in which R₁ and R₂ representindependently of one another a (C₁-C₁₀)alkyl group, a (C₂-C₆)cycloalkylgroup, an aryl group, an aryl(C₁-C₁₀)alkyl group, or a heterocyclicgroup, R₃ and R₄ represent independently of one another a hydrogen atom,a (C₁-C₁₀)alkyl group, an aryl group or an aryl(C₁-C₁₀)alkyl group, andY represents a reactive function which can be activated by clickchemistry, selected from the group comprising the azides, alkynes,cycloalkynes and conjugated dienes, as a diagnostic reagent.
 9. Kit forthe implementation of a coupling and bioconjugation method for couplinga biomolecule and a target molecule selected from the group comprising amolecule of biological interest, a naturel or synthetic polymer, and areactive surface which comprises at least one compound according toclaim
 1. 10. Method for the separation, detection and/orcharacterization of at least one molecule of interest potentiallypresent in a medium, characterized in that it comprises at least onestep of utilizing a kit according to claim 9 or at least one step ofutilizing a compound of formula (I)

in which R₁ and R₂ represent independently of one another a(C₁-C₁₀)alkyl group, a (C₂-C₆)cycloalkyl group, an aryl group, anaryl(C₁-C₁₀)alkyl group, or a heterocyclic group, R₃ and R₄ representindependently of one another a hydrogen atom, a (C₁-C₁₀)alkyl group, anaryl group or an aryl(C₁-C₁₀)alkyl group, and Y represents a reactivefunction which can be activated by click chemistry, selected from thegroup comprising the azides, alkynes, cycloalkynes and conjugateddienes.
 11. Composition comprising a compound according to claim 1 incombination with an aqueous or organic medium.